Composition for producing tagatose and method of producing tagatose using the same

ABSTRACT

Provided are a composition for producing tagatose, comprising fructose-6-phosphate 4-epimerase, and a method of producing tagatose using the same.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present disclosure relates to a composition for producing tagatose,comprising fructose-6-phosphate 4-epimerase, and a method of producingtagatose using the same.

2. Description of the Related Art

Tagatose is a natural sweetener, which is present in a small amount infoods such as milk, cheese, cacao, etc., and in sweet fruits such asapples and mandarin, and has physical properties similar to sucrose.Tagatose has a calorie value of 1.5 kcal/g which is one third that ofsucrose, and a glycemic index (GI) of 3 which is 5% that of sucrose.Tagatose has a sweet taste similar to that of sucrose and various healthbenefits. In this regard, tagatose can be used as an alternativesweetener capable of satisfying both taste and health when applied to awide variety of products.

Conventionally known methods of producing tagatose include a chemicalmethod (a catalytic reaction) and a biological method (an isomerizationenzyme reaction) of using galactose as a main raw material (see PCT WO2006/058092, Korean Patent Nos. 10-0964091 and 10-1368731). However, theprice of lactose which is a basic raw material of galactose in the knownproduction methods is unstable, depending on produced amounts, supply,and demand of raw milk and lactose in global markets, etc. Thus, thereis a limitation in the stable supply of the raw material for tagatoseproduction. Therefore, a new method capable of producing tagatose from acommonly used sugar (sucrose, glucose, fructose, etc.) as a raw materialhas been needed and studied, and the above-mentioned documents disclosea method of producing galactose, psicose, and tagatose from glucose,galactose, and fructose, respectively (Korean Patent Nos. 10-744479,10-1057873, and 10-1550796).

Meanwhile, tagatose-6-phosphate kinase (EC 2.7.1.144) is known toproduce ADP and D-tagatose 1,6-biphosphate from ATP andD-tagatose-6-phosphate as a substrate, as in the following [ReactionScheme 1]. However, there have been no studies regarding whether thetagatose-6-phosphate kinase has activity to convert fructose-6-phosphateinto tagatose-6-phosphate.

ATP+D-tagatose 6-phosphate<=>ADP+D-tagatose 1,6-biphosphate  [ReactionScheme 1]

Under this background, the present inventors have conducted extensivestudies to develop a method capable of increasing a conversion rate intotagatose while using an economic raw material, and as a result, theyfound that tagatose-6-phosphate kinase (EC 2.7.1.144) has the ability toconvert fructose-6-phosphate into tagatose-6-phosphate, therebycompleting the present disclosure.

Accordingly, glucose or starch may be used as a raw material tosequentially produce glucose-1-phosphate and glucose-6-phosphate, andthen tagatose-6-phosphate kinase of the present disclosure may be usedto convert glucose-6-phosphate into tagatose-6-phosphate, andtagatose-6-phosphate phosphatase which performs an irreversible reactionpathway may be used to produce tagatose while remarkably increasing aconversion rate of glucose or starch into tagatose.

SUMMARY OF THE INVENTION

An object of the present disclosure is to provide a composition usefulfor the production of tagatose-6-phosphate, comprisingtagatose-6-phosphate kinase, a microorganism expressing thetagatose-6-phosphate kinase, or a culture of the microorganism.

Another object of the present disclosure is to provide a compositionuseful for the production of tagatose, comprising tagatose-6-phosphatekinase, a microorganism expressing the tagatose-6-phosphate kinase, or aculture of the microorganism; and tagatose-6-phosphate phosphatase, themicroorganism expressing the tagatose-6-phosphate phosphatase, or aculture of the microorganism.

Another object of the present disclosure is to provide a method ofproducing tagatose-6-phosphate, comprising convertingfructose-6-phosphate into tagatose-6-phosphate by contactingfructose-6-phosphate with tagatose-6-phosphate kinase, a microorganismexpressing the tagatose-6-phosphate kinase, or a culture of themicroorganism. The method further comprises convertingtagatose-6-phosphate into tagatose by contacting tagatose-6-phosphatewith tagatose-6-phosphate phosphatase, a microorganism expressing thetagatose-6-phosphate phosphatase, or a culture of the microorganism.

Other objects and advantages of the present disclosure will be describedin more detail with reference to the following description along withthe accompanying claims and drawings. Since contents that are notdescribed in the present specification may be sufficiently recognizedand inferred by those skilled in the art or similar art, a descriptionthereof will be omitted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a result of protein electrophoresis (SDS-PAGE) to analyzemolecular weights of enzymes used in the production pathways of tagatosefrom starch, sucrose or glucose, wherein M represents a protein sizeladder (size marker, Bio-RAD, USA);

FIG. 2 is a result of HPLC chromatography showing that CJ_AN1_F6P4Ewhich is an enzyme of one embodiment of the present disclosure hasfructose-6-phosphate 4-epimerase activity;

FIG. 3 is a result of HPLC chromatography showing that CJ_DT_F6P4E whichis an enzyme of one embodiment of the present disclosure hasfructose-6-phosphate 4-epimerase activity;

FIG. 4 is a result of HPLC chromatography showing that CJ_AB_F6P4E whichis an enzyme of one embodiment of the present disclosure hasfructose-6-phosphate 4-epimerase activity;

FIG. 5 is a result of HPLC chromatography showing that treatment offructose-6-phosphate with tagatose-6-phosphate kinase (CJ_DT_F6P4E) andCJ_T4_T6PP converts fructose-6-phosphate into tagatose in one embodimentof the present disclosure;

FIG. 6 is a result of HPLC chromatography showing that when all of theenzymes involved in the production pathway of tagatose from maltodextrinwere added at the same time, tagatose was produced by complex enzymereactions, wherein CJ_AN1_F6P4E was used as tagatose-6-phosphate kinase;and

FIG. 7 is a result of HPLC chromatography showing that T4 which is anenzyme of the present disclosure has tagatose-6-phosphate phosphataseactivity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Hereinafter, the present disclosure will be described in detail asfollows. Meanwhile, each description and embodiment disclosed in thisdisclosure may be applied to other descriptions and embodiments tocommon things. Further, all combinations of various elements disclosedin this disclosure fall within the scope of the present disclosure.Further, the scope of the present disclosure is not limited by thespecific description described below.

To achieve one object of the present disclosure, an aspect of thepresent disclosure provides a composition for producingtagatose-6-phosphate, comprising tagatose-6-phosphate kinase, amicroorganism expressing the tagatose-6-phosphate kinase, or a cultureof the microorganism.

The tagatose-6-phosphate kinase (EC 2.7.1.144) is known to produce ADPand D-tagatose 1,6-biphosphate from ATP and D-tagatose-6-phosphate as asubstrate. For example, the tagatose-6-phosphate kinase may be any onewithout limitation as long as it is able to produce tagatose-6-phosphatefrom fructose-6-phosphate as a substrate.

The tagatose-6-phosphate kinase may be a polypeptide consisting of anamino acid sequence of SEQ ID NO: 1, 3, or 5, or comprise a polypeptidehaving at least 80%, 90%, 95%, 97%, or 99% homology with the amino acidsequence of SEQ ID NO: 1, 3, or 5. It is apparent that a polypeptidehaving the homology and an amino acid sequence exhibiting the efficacy(i.e., fructose-6-phosphate C4-epimerization activity to convertfructose-6-phosphate into tagatose-6-phosphate by epimerizingfructose-6-phosphate at C4 position of fructose) corresponding to theprotein consisting of the amino acid sequence of SEQ ID NO: 1, 3, or 5is also included in the scope of the present disclosure, although it hasan amino acid sequence, of which a partial sequence is deleted,modified, substituted, or added. Further, a probe which may be producedfrom the known nucleotide sequence, for example, a polypeptide encodedby a polynucleotide which is hybridizable with a complementary sequenceto all or a part of a nucleotide sequence encoding the polypeptide understringent conditions may be also included without limitation, as long asit has the fructose-6-phosphate C4-epimerization activity. Further, thecomposition may comprise one or more of tagatose-6-phosphate kinaseconsisting of the amino acid sequence of SEQ ID NO: 1, 3, or 5.

The present disclosure revealed that the ‘tagatose-6-phosphate kinase’exhibits the fructose-6-phosphate 4-epimerization activity to convertfructose-6-phosphate into tagatose-6-phosphate by epimerizingfructose-6-phosphate at C4 position. In the present disclosure,therefore, the ‘tagatose-6-phosphate kinase’ may be used interchangeablywith ‘fructose-6-phosphate C4 epimerase’.

As used herein, the term “stringent conditions” mean conditions underwhich specific hybridization between polynucleotides is allowed. Theseconditions depend on the length of the polynucleotide and the degree ofcomplementation, and variables are well known in the art, andspecifically described in a literature (e.g., J. Sambrook et al.,infra). The stringent conditions may include, for example, conditionsunder which genes having high homology, 80% or higher homology, 90% orhigher homology, 95% or higher homology, 97% or higher homology, 99% orhigher homology are hybridized with each other and genes having homologylower than the above homology are not hybridized with each other, orordinary washing conditions of Southern hybridization, i.e., washingonce, specifically, twice or three times at a salt concentration and atemperature corresponding to 60° C., 1×SSC, 0.1% SDS, specifically, 60°C., 0.1×SSC, 0.1% SDS, and more specifically 68° C., 0.1×SSC, 0.1% SDS.The probe used in the hybridization may be a part of a complementarysequence of the nucleotide sequence. Such a probe may be produced by PCRusing oligonucleotides produced based on the known sequence as primersand a DNA fragment comprising these nucleotide sequences as a template.Further, those skilled in the art may adjust the temperature and thesalt concentration of the washing solution according to factors such asthe length of the probe, if necessary.

As used herein, the term “homology” refers to a percentage of identitybetween two polypeptide moieties. Sequence correspondence from onemoiety to another may be determined by a known technique in the art. Forexample, the homology may be determined by directly aligning thesequence information of two polypeptide molecules, e.g., parameters suchas score, identity, and similarity, etc., using a computer program thatis readily available and capable of aligning sequence information (e.g.,BLAST 2.0). Additionally, the homology between polynucleotides may bedetermined by hybridizing the polynucleotides under a condition forforming a stable double-strand in the homologous regions followed bydigesting the hybridized strand by a single-strand-specific nuclease todetermine the size of digested fragments.

In one embodiment, the tagatose-6-phosphate kinase of the presentdisclosure may be an enzyme derived from a heat-resistant microorganism,for example, an enzyme derived from Anaerolinea sp. or a variantthereof, or an enzyme derived from the genus of Dictyoglomus or avariant thereof, and specifically, an enzyme derived from Anaerolineathermophila, Anaerolineae bacterium, or Dictyoglomus thermophilum, or avariant thereof, but is not limited thereto.

The fructose-6-phosphate 4-epimerase of the present disclosure or avariant thereof is characterized by converting D-fructose-6-phosphateinto D-tagatose-6-phosphate by epimerizing D-fructose-6-phosphate at C4position. An enzyme which is known to have tagatose-6-phosphate kinaseactivity may be used as the fructose-6-phosphate 4-epimerase of thepresent disclosure, and the tagatose-6-phosphate kinase was known toproduce D-tagatose 1,6-biphosphate from D-tagatose-6-phosphate as asubstrate. The present disclosure newly revealed that thetagatose-6-phosphate kinase has the fructose-6-phosphate-4-epimeraseactivity. Accordingly, one embodiment of the present disclosure relatesto novel use of the tagatose-6-phosphate kinase including using thetagatose-6-phosphate kinase as the fructose-6-phosphate-4-epimerase inthe production of tagatose-6-phosphate from fructose-6-phosphate.Further, one embodiment of the present disclosure relates to a method ofproducing tagatose-6-phosphate from fructose-6-phosphate using thetagatose-6-phosphate kinase as the fructose-6-phosphate-4-epimerase.

In one embodiment, the fructose-6-phosphate 4-epimerase of the presentdisclosure may be an enzyme having high heat resistance. Specifically,the fructose-6-phosphate-4-epimerase of the present disclosure mayexhibit 50% to 100%, 60% to 100%, 70% to 100%, or 75% to 100% of itsmaximum activity at 50° C. to 70° C. More specifically, thefructose-6-phosphate-4-epimerase of the present disclosure may exhibit80% to 100% or 85% to 100% of its maximum activity at 55° C. to 65° C.,60° C. to 70° C., 55° C., 60° C., or 70° C.

Furthermore, the fructose-6-phosphate-4-epimerase consisting of theamino acid sequence of SEQ ID NO: 1, 3, or 5 may be, but is not limitedto, encoded by a nucleotide sequence of SEQ ID NO: 2, 4, or 6,respectively.

The fructose-6-phosphate 4-epimerase of the present disclosure or avariant thereof may be obtained by transforming a microorganism such asE. coli with DNA expressing the enzyme of the present disclosure or thevariant thereof, e.g., SEQ ID NO: 2, 4, or 6, culturing themicroorganism to obtain a culture, disrupting the culture, and thenperforming purification using a column, etc. The microorganism fortransformation may include Corynebacterium glutamicum, Aspergillusoryzae, or Bacillus subtilis, etc., in addition to Escherichia coli. Ina specific embodiment, the transformed microorganism may be E. coliBL21(DE3)/pBT7-C-His-an1, E. coli BL21(DE3)/pBT7-C-His-CJ_AB_F6P4E, orE. coli BL21 (DE3)/pBT7-C-His-CJ_DT_F6P4E. E. coliBL21(DE3)/pBT7-C-His-an1 was deposited on Mar. 20, 2017 with AccessionNo. KCCM11996P, E. coli BL21 (DE3)/pBT7-C-His-CJ_DT_F6P4E was depositedon Sep. 13, 2017 with Accession No. KCCM12110P, and E. coliBL21(DE3)/pBT7-C-His-CJ_AB_F6P4E was deposited on Aug. 11, 2017 withAccession No. KCCM12093P at the Korean Culture Center of Microorganisms.

The fructose-6-phosphate-4-epimerase used in the present disclosure maybe provided by using a nucleic acid encoding the same.

As used herein, the term “nucleic acid” means that it encompasses DNA orRNA molecules, wherein nucleotides which are basic constituent units inthe nucleic acid may include not only natural nucleotides but alsoanalogues with modification of sugar or base (see: Scheit, NucleotideAnalogs, John Wiley, New York (1980); Uhlman and Peyman, ChemicalReviews, 90:543-584(1990)).

The nucleic acid of the present disclosure may be a nucleic acidencoding the polypeptide consisting of the amino acid sequence of SEQ IDNO: 1, 3, or 5 of the present disclosure or a nucleic acid encoding apolypeptide having at least 80%, 90%, 95%, 97% or 99% homology with thefructose-6-phosphate-4-epimerase of the present disclosure and thefructose-6-phosphate-4-epimerase activity. For example, the nucleic acidencoding the fructose-6-phosphate-4-epimerase consisting of the aminoacid sequence of SEQ ID NO: 1 may be a nucleic acid having at least 80%,90%, 95%, 97%, 99% or 100% homology with the nucleotide sequence of SEQID NO: 2. Further, the nucleic acid encoding thefructose-6-phosphate-4-epimerase consisting of the amino acid sequenceof SEQ ID NO: 3 or 5 may be a nucleic acid having at least 80%, 90%,95%, 97%, 99% or 100% homology with the nucleotide sequence of SEQ IDNO: 4 or 6 corresponding thereto, respectively. It is also apparent thatthe nucleic acid of the present disclosure may include a nucleic acidwhich is translated into the fructose-6-phosphate-4-epimerase of thepresent disclosure due to codon degeneracy and a nucleic acid whichhybridizes with a nucleic acid consisting of a nucleotide sequencecomplementary to the nucleotide sequence of SEQ ID NO: 2, 4, or 6 understringent conditions and encodes the polypeptide having thefructose-6-phosphate-4-epimerase activity of the present disclosure.

The microorganism expressing the fructose-6-phosphate-4-epimerase whichmay be used in the present disclosure may be a microorganism comprisinga recombinant vector comprising the nucleic acid.

The vector may be operably linked to the nucleic acid of the presentdisclosure. As used herein, the term “operably linked” means that anucleotide expression regulatory sequence and a nucleotide sequenceencoding a targeted protein are operably linked to each other to performthe general functions, thereby affecting expression of the encodingnucleotide sequence. The operable linkage to the vector may be producedusing a genetic recombination technology known in the art, and thesite-specific DNA cleavage and linkage may be produced using restrictionenzymes and ligases known in the art.

As used herein, the term “vector” refers to any mediator for cloningand/or transferring of bases into an organism, such as a host cell. Thevector may be a replicon that is able to bring the replication ofcombined fragments in which different DNA fragments are combined. Here,the term “replicon” refers to any genetic unit (e.g., plasmid, phage,cosmid, chromosome, virus) which functions as a self-unit of DNAreplication in vivo, i.e., which is able to be replicated byself-regulation. As used herein, the term “vector” may include viral andnon-viral mediators for introducing the bases into the organism, e.g., ahost cell, in vitro, ex vivo, or in vivo, and may also include aminicircular DNA, a transposon such as Sleeping Beauty (Izsvak et al. J.Mol. Biol. 302:93-102 (2000)), or an artificial chromosome. Examples ofthe vector commonly used may include natural or recombinant plasmids,cosmids, viruses, and bacteriophages. For example, as a phage vector orcosmid vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11,Charon4A, and Charon21A, etc., may be used; and as a plasmid vector,those based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET, etc.,may be used. The vectors that may be used in the present disclosure arenot particularly limited, but any known expression vector may be used.Further, the vector may be a recombinant vector characterized by furthercomprising various antibiotic resistance genes. As used herein, the term“antibiotic resistance gene” refers to a gene having resistance againstan antibiotic, and a cell having this gene survives in an environmenttreated with the corresponding antibiotic. Thus, the antibioticresistance gene is used as a selectable marker during production of alarge amount of plasmids in E. coli. The antibiotic resistance gene inthe present disclosure is not a factor that greatly influencesexpression efficiency according to optimal combinations of vectors whichis a key technology of the present disclosure, and thus an antibioticresistance gene that is generally used as a selectable marker may beused without limitation. Specific examples may include a resistance geneagainst ampicilin, tetracyclin, kanamycin, chloroamphenicol,streptomycin, or neomycin etc.

The microorganism expressing the fructose-6-phosphate-4-epimerase whichmay be used in the present disclosure may be obtained by a method ofintroducing the vector comprising the nucleic acid encoding the enzymeinto a host cell, and a method of transforming the vector may beincluded as long as it is able to introduce the nucleic acid into thecell. An appropriate standard technique known in the art may be selectedand performed. Electroporation, calcium phosphate co-precipitation,retroviral infection, microinjection, a DEAE-dextran method, a cationicliposome method, and a heat shock method may be included, but is notlimited thereto.

As long as the transformed gene may be expressed in the host cell, itmay be inserted into the chromosome of the host cell, and it may existextrachromosomally. Further, the gene includes DNA and RNA as apolynucleotide encoding a polypeptide, and any form may be used withoutlimitation, as long as it may be introduced into the host cell andexpressed therein. For example, the gene may be introduced into the hostcell in the form of an expression cassette, which is a polynucleotideconstruct comprising all elements required for its autonomousexpression. Commonly, the expression cassette may comprise a promoteroperably linked to the gene, transcriptional termination signals,ribosome binding sites, and translation termination signals. Theexpression cassette may be in the form of a self-replicable expressionvector. Also, the gene as it is or in the form of a polynucleotideconstruct may be introduced into the host cell and operably linked tosequences required for expression in the host cell.

The microorganism of the present disclosure may include either aprokaryotic microorganism and a eukaryotic microorganism, as long as itis a microorganism capable of producing thefructose-6-phosphate-4-epimerase of the present disclosure by comprisingthe nucleic acid of the present disclosure or the recombinant vector ofthe present disclosure. For example, the microorganism may includemicroorganism strains belonging to the genus Escherichia, the genusErwinia, the genus Serratia, the genus Providencia, the genusCorynebacterium, and the genus Brevibacterium, and specifically, it maybe E. coli or Corynebacterium glutamicum, but is not limited thereto.Specific examples of the microorganism may include E. coliBL21(DE3)/pBT7-C-His-an1, E. coli BL21(DE3)/pBT7-C-His-CJ_AB_F6P4E, orE. coli BL21(DE3)/pBT7-C-His-CJ_DT_F6P4E, etc.

The microorganism of the present disclosure may include anymicroorganism capable of expressing the fructose-6-phosphate-4-epimeraseof the present disclosure or related enzymes according to various knownmethods, in addition to introduction of the nucleic acid or the vector.

The culture of the microorganism of the present disclosure may beproduced by culturing, in a medium, the microorganism expressing thetagatose-6-phosphate kinase of the present disclosure or relatedenzymes.

As used herein, the term “culturing” means that the microorganism isallowed to grow under appropriately controlled environmental conditions.The culturing process of the present disclosure may be carried outaccording to an appropriate medium and culture conditions known in theart. The culturing process may be easily adjusted by those skilled inthe art according to the strain to be selected. The step of culturingthe microorganism may be, but is not particularly limited to, carriedout by a known batch process, a continuous process, a fed batch processetc. With regard to the culture conditions, a proper pH (e.g., pH 5 to9, specifically pH 7 to 9) may be adjusted using a basic compound (e.g.,sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound(e.g., phosphoric acid or sulfuric acid), but is not particularlylimited thereto. Additionally, an antifoaming agent such as fatty acidpolyglycol ester may be added during the culturing process to preventfoam generation. Additionally, oxygen or an oxygen-containing gas may beinjected into the culture in order to maintain an aerobic state of theculture; or nitrogen, hydrogen, or carbon dioxide gas may be injectedwithout the injection of a gas in order to maintain an anaerobic ormicroaerobic state of the culture. The culture temperature may bemaintained from 25° C. to 40° C., and specifically, from 30° C. to 37°C., but is not limited thereto. The culturing may be continued until thedesired amount of useful materials is obtained, and specifically forabout 0.5 hours to about 60 hours, but is not limited thereto.Furthermore, the culture medium to be used may comprise, as carbonsources, sugars and carbohydrates (e.g., glucose, sucrose, lactose,fructose, maltose, molasses, starch, and cellulose), oils and fats(e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fattyacids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols(e.g., glycerol and ethanol), and organic acids (e.g., acetic acid) etc.These substances may be used individually or in a mixture, but are notlimited thereto. Nitrogen sources may include nitrogen-containingorganic compounds (e.g., peptone, yeast extract, meat extract, maltextract, corn steep liquor, soybean meal, and urea) or inorganiccompounds (e.g., ammonium sulfate, ammonium chloride, ammoniumphosphate, ammonium carbonate, and ammonium nitrate) etc. These nitrogensources may also be used individually or in a mixture, but are notlimited thereto. Phosphorus sources may include potassium dihydrogenphosphate, dipotassium hydrogen phosphate, the correspondingsodium-containing salts etc. These nitrogen sources may also be usedindividually or in a mixture, but are not limited thereto. The culturemedium may include essential growth stimulators, such as metal salts(e.g., magnesium sulfate or iron sulfate), amino acids, and vitamins.

The composition for producing tagatose-6-phosphate of the presentdisclosure may further comprise fructose-6-phosphate, and thefructose-6-phosphate may be converted into tagatose-6-phosphate by thefructose-6-phosphate-4-epimerase of the present disclosure.

Another aspect of the present disclosure provides a composition forproducing tagatose, comprising tagatose-6-phosphate kinase, amicroorganism expressing the tagatose-6-phosphate kinase, or a cultureof the microorganism; and tagatose-6-phosphate phosphatase, themicroorganism expressing the tagatose-6-phosphate phosphatase, or aculture of the microorganism. The above description of thetagatose-6-phosphate kinase, the microorganism expressing thetagatose-6-phosphate kinase, or the culture of the microorganism in thisaspect may be also applied to those above-described aspect.

The tagatose-6-phosphate phosphatase of the present disclosure maycomprise any protein without limitation, as long as it has activity toconvert tagatose-6-phosphate into tagatose by eliminating a phosphategroup of tagatose-6-phosphate. The tagatose-6-phosphate phosphatase ofthe present disclosure may be an enzyme derived from a heat-resistantmicroorganism, for example, an enzyme derived from Thermotoga sp. or avariant thereof, specifically, an enzyme derived from Thermotogamaritima or a variant thereof.

According to one embodiment of the present disclosure, thetagatose-6-phosphate phosphatase of the present disclosure may be aprotein which consists of an amino acid sequence of SEQ ID NO: 7, orconsists of a sequence having a genetic homology of 70%, 75%, 80%, 85%,90%, 95%, 97%, 99%, or 100% thereto, or a sequence having a genetichomology within the range determined by any two values of the abovevalues. According to one embodiment of the present disclosure, thetagatose-6-phosphate phosphatase consisting of the amino acid sequenceof SEQ ID NO: 7 of the present disclosure may be encoded by a nucleotidesequence of SEQ ID NO: 8.

The composition for producing tagatose of the present disclosure mayfurther comprise glucose-6-phosphate isomerase, a microorganismexpressing the glucose-6-phosphate isomerase, or a culture of themicroorganism. In the presence of the enzyme, glucose-6-phosphate may beisomerized to produce fructose-6-phosphate. Theglucose-6-phosphate-isomerase of the present disclosure may include anyprotein without limitation, as long as it has activity to isomerizeglucose-6-phosphate into fructose-6-phosphate. Theglucose-6-phosphate-isomerase of the present disclosure may be an enzymederived from a heat-resistant microorganism, for example, an enzymederived from Thermotoga sp. or a variant thereof, specifically, anenzyme derived from Thermotoga maritima or a variant thereof. Accordingto one embodiment of the present disclosure, theglucose-6-phosphate-isomerase of the present disclosure may be a proteinwhich consists of an amino acid sequence of SEQ ID NO: 9, or consists ofa sequence having a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%,97%, 99%, or 100% thereto, or a sequence having a homology within therange determined by any two values of the above values. According to oneembodiment of the present disclosure, the glucose-6-phosphate-isomeraseconsisting of the amino acid sequence of SEQ ID NO: 9 of the presentdisclosure may be encoded by a nucleotide sequence of SEQ ID NO: 10.

The composition for producing tagatose of the present disclosure mayfurther comprise phosphoglucomutase, a microorganism expressing thephosphoglucomutase, or a culture of the microorganism, and the enzymemay catalyze a reversible reaction of converting glucose-1-phosphateinto glucose-6-phosphate or converting glucose-6-phosphate intoglucose-1-phosphate. The phosphoglucomutase of the present disclosuremay include any protein without limitation, as long as it has activityto convert glucose-1-phosphate into glucose-6-phosphate or to convertglucose-6-phosphate into glucose-1-phosphate. The phosphoglucomutase ofthe present disclosure may be an enzyme derived from a heat-resistantmicroorganism, for example, an enzyme derived from Thermotoga sp. or avariant thereof, specifically, an enzyme derived from Thermotoganeapolitana or a variant thereof. According to one embodiment of thepresent disclosure, the phosphoglucomutase of the present disclosure maybe a protein which consists of an amino acid sequence of SEQ ID NO: 11,or consists of a sequence having a genetic homology of 70%, 75%, 80%,85%, 90%, 95%, 97%, 99%, or 100% thereto, or within the range determinedby any two values of the above values. According to one embodiment ofthe present disclosure, the phosphoglucomutase consisting of the aminoacid sequence of SEQ ID NO: 11 of the present disclosure may be encodedby a nucleotide sequence of SEQ ID NO: 12.

The composition for producing tagatose of the present disclosure mayfurther comprise glucokinase, a microorganism expressing theglucokinase, or a culture of the microorganism. The glucokinase of thepresent disclosure may include any protein without limitation, as longas it has activity to phosphorylate glucose. The glucokinase of thepresent disclosure may be an enzyme derived from a heat-resistantmicroorganism, for example, an enzyme derived from Deinococcus sp. orAnaerolinea sp., or a variant thereof, specifically, an enzyme derivedfrom Deinococcus geothermalis, Anaerolinea thermophila, or a variantthereof.

The glucokinase of the present disclosure may include any proteinwithout limitation, as long as it has activity to convert glucose intoglucose-6-phosphate. Specifically, the glucokinase of the presentdisclosure may be a phosphate-dependent glucokinase. According to oneembodiment of the present disclosure, the glucokinase of the presentdisclosure may be a protein which consists of an amino acid sequence ofSEQ ID NO: 13 or 15, or consists of a sequence having a genetic homologyof 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% thereto, or withinthe range determined by any two values of the above values. According toone embodiment of the present disclosure, the glucokinase consisting ofthe amino acid sequence of SEQ ID NO: 13 of the present disclosure maybe encoded by a nucleotide sequence of SEQ ID NO: 14, and theglucokinase consisting of the amino acid sequence of SEQ ID NO: 15 ofthe present disclosure may be encoded by a nucleotide sequence of SEQ IDNO: 16.

The composition for producing tagatose of the present disclosure mayfurther comprise α-glucan phosphorylase, starch phosphorylase,maltodextrin phosphorylase, or sucrose phosphorylase, a microorganismexpressing the α-glucan phosphorylase, starch phosphorylase,maltodextrin phosphorylase, or sucrose phosphorylase, or a culture ofthe microorganism. The phosphorylase may include any protein withoutlimitation, as long as it has activity to convert starch, maltodextrin,or sucrose into glucose-1-phosphate. The phosphorylase may be an enzymederived from a heat-resistant microorganism, for example, an enzymederived from Thermotoga sp. or a variant thereof, specifically, anenzyme derived from Thermotoga neapolitana or a variant thereof. Thephosphorylase of the present disclosure may be a protein which consistsof an amino acid sequence of SEQ ID NO: 17, or consists of a sequencehaving a genetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or100% thereto, or within the range determined by any two values of theabove values. According to one embodiment of the present disclosure, thephosphorylase composed of the amino acid sequence of SEQ ID NO: 17 ofthe present disclosure may be encoded by a nucleotide sequence of SEQ IDNO: 18.

The composition for producing tagatose of the present disclosure mayfurther comprise α-amylase, pullulanase, glucoamylase, sucrase, orisoamylase; a microorganism expressing the amylase, pullulanase,glucoamylase, sucrase, or isoamylase; or a culture of the microorganismexpressing the amylase, pullulanase, glucoamylase, sucrase, orisoamylase.

The composition for producing tagatose of the present disclosure maycomprise two or more enzymes of the above-described enzymes which may beused in the production of tagatose or transformants thereofindividually, or a transformant transformed with nucleotides encodingthe two or more enzymes.

The composition for producing tagatose of the present disclosure mayfurther comprise 4-α-glucanotransferase, a microorganism expressing the4-α-glucanotransferase, or a culture of the microorganism expressing the4-α-glucanotransferase. The 4-α-glucanotransferase of the presentdisclosure may include any protein without limitation, as long as it hasactivity to convert glucose into starch, maltodextrin, or sucrose. The4-α-glucanotransferase of the present disclosure may be an enzymederived from a heat-resistant microorganism, for example, an enzymederived from Thermotoga sp. or a variant thereof, specifically, anenzyme derived from Thermotoga maritima or a variant thereof. Accordingto one embodiment of the present disclosure, the 4-α-glucanotransferaseof the present disclosure may be a protein which consists of an aminoacid sequence of SEQ ID NO: 19, or consists of a sequence having agenetic homology of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%thereto, or within the range determined by any two values of the abovevalues. According to one embodiment of the present disclosure, the4-α-glucanotransferase consisting of the amino acid sequence of SEQ IDNO: 19 of the present disclosure may be encoded by a nucleotide sequenceof SEQ ID NO: 20.

Examples of the microorganisms which may be used in the above-describedembodiments may include E. coli BL21 (DE3)/pET21a-CJ_ct1, E. coli BL21(DE3)/pET21a-CJ_ct2, E. coli BL21 (DE3)/pET21a-CJ_tn1, E. coli BL21(DE3)/pET21a-CJ_tn2, and E. coli BL21 (DE3)/pET21a-CJ_t4, etc. Therecombinant microorganisms were deposited at Korean Culture Center ofMicroorganisms on Mar. 20, 2017 with Accession Nos. KCCM11990P (E. coliBL21(DE3)/pET21a-CJ_ct1), KCCM11991P (E. coli BL21(DE3)/pET21a-CJ_ct2),KCCM11992P (E. coli BL21 (DE3)/pET21a-CJ_tn1), KCCM11993P (E. coliBL21(DE3)/pET21a-CJ_tn2), KCCM11994P (E. coli BL21 (DE3)/pET21a-CJ_t4),respectively.

The composition for producing tagatose of the present disclosure mayfurther comprise a substance, a component, or a compositioncorresponding to each substrate of the above-described enzymes.

The composition for producing tagatose of the present disclosure mayfurther comprise any suitable excipient commonly used in thecorresponding composition for producing tagatose. The excipient mayinclude, for example, a preservative, a wetting agent, a dispersingagent, a suspending agent, a buffer, a stabilizing agent, or an isotonicagent, etc., but is not limited thereto.

The composition for producing tagatose of the present disclosure mayfurther comprise a metal. In one embodiment, the metal of the presentdisclosure may be a metal comprising a divalent cation. Specifically,the metal of the present disclosure may be one or more selected from thegroup consisting of nickel, cobalt, aluminum, magnesium (Mg), andmanganese (Mn). More specifically, the metal of the present disclosuremay be a metal ion or a metal salt, and much more specifically, themetal salt may be one or more selected from the group consisting ofNiSO₄, MgSO₄, MgCl₂, NiCl₂, CoCl₂, CoSO₄, MnCl₂, and MnSO₄.

Another aspect of the present disclosure relates to a method ofproducing tagatose-6-phosphate, comprising convertingfructose-6-phosphate into tagatose-6-phosphate by contactingfructose-6-phosphate with tagatose-biphosphate aldolase, themicroorganism expressing the tagatose-biphosphate aldolase, or theculture of the microorganism.

The description of the composition for producing tagatose-6-phosphatemay be also applied to the composition for producing tagatose.

Another aspect of the present disclosure relates to a method ofproducing tagatose-6-phosphate, comprising nverting fructose-6-phosphateinto tagatose-6-phosphate by contacting fructose-6-phosphate withtagatose-6-phosphate kinase, the microorganism expressing thetagatose-6-phosphate kinase, or the culture of the microorganism. Themethod may further comprise converting tagatose-6-phosphate intotagatose by contacting tagatose-6-phosphate with tagatose-6-phosphatephosphatase, the microorganism expressing the tagatose-6-phosphatephosphatase, or the culture of the microorganism.

The method of the present disclosure may further comprise convertingglucose-6-phosphate into fructose-6-phosphate by contactingglucose-6-phosphate with the glucose-6-phosphate-isomerase of thepresent disclosure, the microorganism expressing theglucose-6-phosphate-isomerase, or the culture of the microorganismexpressing the glucose-6-phosphate-isomerase.

The method of the present disclosure may further comprise convertingglucose-1-phosphate into glucose-6-phosphate by contactingglucose-1-phosphate with the phosphoglucomutase of the presentdisclosure, the microorganism expressing the phosphoglucomutase, or theculture of the microorganism expressing the phosphoglucomutase.

The method of the present disclosure may further comprise convertingglucose into glucose-6-phosphate by contacting glucose with theglucokinase of the present disclosure, the microorganism expressing theglucokinase, or the culture of the microorganism expressing theglucokinase.

The method of the present disclosure may further comprise convertingstarch, maltodextrin, or sucrose into glucose-1-phosphate by contactingstarch, maltodextrin, sucrose, or a combination thereof with theα-glucan phosphorylase, starch phosphorylase, maltodextrinphosphorylase, sucrose phosphorylase of the present disclosure, themicroorganism expressing the phosphorylase, or the culture of themicroorganism expressing the phosphorylase.

The method of the present disclosure may further comprise convertingstarch, maltodextrin, or sucrose into glucose by contacting starch,maltodextrin, sucrose, or a combination thereof with the α-amylase,pullulanase, glucoamylase, sucrase, or isoamylase; the microorganismexpressing the α-amylase, pullulanase, glucoamylase, sucrase, orisoamylase; or the culture of the microorganism expressing theα-amylase, pullulanase, glucoamylase, sucrase, or isoamylase.

The method of the present disclosure may further comprise convertingglucose into starch, maltodextrin, or sucrose by contacting glucose withthe 4-α-glucanotransferase of the present disclosure, the microorganismexpressing the 4-α-glucanotransferase, or the culture of themicroorganism expressing the 4-α-glucanotransferase.

Each contacting in the method of the present disclosure may be performedunder conditions of pH 5.0 to pH 9.0, 30° C. to 80° C. and/or for 0.5hours to 48 hours. Specifically, the contacting of the presentdisclosure may be performed under a condition of pH 6.0 to pH 9.0 or pH7.0 to pH 9.0.

Further, the contacting of the present disclosure may be performed undera temperature condition of 35° C. to 80° C., 40° C. to 80° C., 45° C. to80° C., 50° C. to 80° C., 55° C. to 80° C., 60° C. to 80° C., 30° C. to70° C., 35° C. to 70° C., 40° C. to 70° C., 45° C. to 70° C., 50° C. to70° C., 55° C. to 70° C., 60° C. to 70° C., 30° C. to 65° C., 35° C. to65° C., 40° C. to 65° C., 45° C. to 65° C., 50° C. to 65° C., 55° C. to65° C., 30° C. to 60° C., 35° C. to 60° C., 40° C. to 60° C., 45° C. to60° C., 50° C. to 60° C. or 55° C. to 60° C. Furthermore, the contactingof the present disclosure may be performed for 0.5 hours to 36 hours,0.5 hours to 24 hours, 0.5 hours to 12 hours, 0.5 hours to 6 hours, 1hour to 36 hours, 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 6hours, 3 hours to 36 hours, 3 hours to 24 hours, 3 hours to 12 hours, 3hours to 6 hours, 12 hours to 36 hours, or 18 hours to 30 hours.

In one embodiment, the contacting of the present disclosure may beperformed in the presence of a metal, a metal ion, or a metal salt.

Another aspect of the present disclosure relates to a method ofproducing tagatose, comprising contacting the composition for producingtagatose described herein with starch, maltodextrin, sucrose, or acombination thereof; and phosphate.

In a specific embodiment of the present disclosure, a method ofproducing tagatose is provided, comprising: converting glucose intoglucose-6-phosphate by contacting glucose with the glucokinase of thepresent disclosure, the microorganism expressing the glucokinase, or theculture of the microorganism, converting glucose-6-phosphate intofructose-6-phosphate by contacting glucose-6-phosphate with theglucose-6-phosphate-isomerase of the present disclosure, themicroorganism expressing the glucose-6-phosphate-isomerase, or theculture of the microorganism, converting fructose-6-phosphate intotagatose-6-phosphate by contacting fructose-6-phosphate with thefructose-6-phosphate-4-epimerase of the present disclosure, themicroorganism expressing the fructose-6-phosphate-4-epimerase, or theculture of the microorganism, and converting tagatose-6-phosphate intotagatose by contacting tagatose-6-phosphate with thetagatose-6-phosphate phosphatase of the present disclosure, themicroorganism expressing the tagatose-6-phosphate phosphatase, or theculture of the microorganism.

Each conversion reaction may be performed sequentially or in situ in thesame reaction system. In the method, phosphate released fromtagatose-6-phosphate by phosphatase may be used as a substrate of theglucokinase to produce glucose-6-phosphate. Therefore, phosphate is notaccumulated and as a result, a high conversion rate may be obtained.

In the method, glucose may be, for example, produced by convertingstarch, maltodextrin, or sucrose into glucose by contacting starch,maltodextrin, sucrose, or a combination thereof with α-glucanphosphorylase, starch phosphorylase, maltodextrin phosphorylase, sucrosephosphorylase of the present disclosure, the microorganism expressingthe phosphorylase, or the culture of the microorganism expressing thephosphorylase. Therefore, the method according to a specific embodimentmay further comprise converting starch, maltodextrin, or sucrose intoglucose.

In another specific embodiment of the present disclosure, a method ofproducing tagatose is provided, comprising:

converting glucose-1-phosphate into glucose-6-phosphate by contactingglucose-1-phosphate with the phosphoglucomutase of the presentdisclosure, the microorganism expressing the phosphoglucomutase, or theculture of the microorganism,

converting glucose-6-phosphate into fructose-6-phosphate by contactingglucose-6-phosphate with the glucose-6-phosphate-isomerase of thepresent disclosure, the microorganism expressing theglucose-6-phosphate-isomerase, or the culture of the microorganism,

converting fructose-6-phosphate into tagatose-6-phosphate by contactingfructose-6-phosphate with the fructose-6-phosphate-4-epimerase of thepresent disclosure, the microorganism expressing thefructose-6-phosphate-4-epimerase, or the culture of the microorganism,and

converting tagatose-6-phosphate into tagatose by contactingtagatose-6-phosphate with the tagatose-6-phosphate phosphatase of thepresent disclosure, the microorganism expressing thetagatose-6-phosphate phosphatase, or the culture of the microorganism.

Each conversion reaction may be performed sequentially or in situ in thesame reaction system.

In the method, glucose-1-phosphate may be, for example, produced byconverting starch, maltodextrin, or sucrose into glucose-1-phosphate bycontacting starch, maltodextrin, sucrose, or a combination thereof withα-glucan phosphorylase, starch phosphorylase, maltodextrinphosphorylase, sucrose phosphorylase of the present disclosure, themicroorganism expressing the phosphorylase, or the culture of themicroorganism expressing the phosphorylase. Therefore, the methodaccording to a specific embodiment may further comprise convertingstarch, maltodextrin, or sucrose into glucose-1-phosphate. In thisregard, phosphate released from tagatose-6-phosphate by phosphatase maybe used as a substrate of the phosphorylase to produceglucose-1-phosphate. Therefore, phosphate is not accumulated, and as aresult, a high conversion rate may be obtained.

The method may further comprise purifying the produced tagatose. Thepurification in the method is not particularly limited, and a methodcommonly used in the art to which the present disclosure pertains may beused. Non-limiting examples may include chromatography, fractionalcrystallization, and ion purification, etc. The purification method maybe performed only by a single method or by two or more methods. Forexample, the tagatose product may be purified through chromatography,and separation of the sugar by the chromatography may be performed byutilizing a difference in a weak binding force between the sugar to beseparated and a metal ion attached to an ion resin.

In addition, the present disclosure may further comprise performingdecolorization, desalination, or both of decolorization and desalinationbefore or after the purification step of the present disclosure. Byperforming the decolorization and/or desalination, it is possible toobtain a more purified tagatose product without impurities.

Hereinafter, the present disclosure will be described in more detailwith reference to Examples. However, these Examples are provided forbetter understanding, and the disclosure is not intended to be limitedby these Examples.

Example 1: Production of Recombinant Expression Vector and Transformantof Each Enzyme

To provide α-glucan phosphorylase, phosphoglucomutase,glucose-6-phosphate-isomerase, and 4-α-glucanotransferase which areheat-resistant enzymes needed in the production pathway of tagatose ofthe present disclosure, nucleotide sequences expected as the enzymes[the above enzymes are represented by SEQ ID NO: 18(CT1), SEQ ID NO:12(CT2), SEQ ID NO: 10(TN1), SEQ ID NO: 20(TN2), respectively] wereselected from a nucleotide sequence of a thermophile microorganism,Thermotoga neapolitana or Thermotoga maritima, which is registered inGenbank.

Based on the selected nucleotide sequences, forward primers (SEQ ID NO:21: CT1-Fp, SEQ ID NO: 23: CT2-Fp, SEQ ID NO: 25: TN1-Fp, SEQ ID NO: 27:TN2-Fp) and reverse primers (SEQ ID NO: 22: CT1-Rp, SEQ ID NO: 24:CT2-Rp, SEQ ID NO: 26: TN1-Rp, SEQ ID NO: 28: TN2-Rp) were designed andsynthesized, and the gene of each enzyme was amplified by PCR using theabove primers and a genomic DNA of Thermotoga neapolitana as a template.Each amplified gene of the enzymes was inserted into pET21a (Novagen)which is a plasmid vector for E. coli expression using restrictionenzymes, NdeI and XhoI or SalI, thereby producing recombinant expressionvectors designated as pET21a-CJ_ct1, pET21a-CJ_ct2, pET21a-CJ_tn1,pET21a-CJ_tn2, respectively.

Each of the expression vectors was transformed into E. coli BL21(DE3)according to a common transformation method (see Sambrook et al. 19891,thereby producing transformants (transformed microorganisms) designatedas E. coli BL21 (DE3)/pET21a-CJ_ct1, E. coli BL21 (DE3)/pET21a-CJ_ct2,E. coli BL21 (DE3)/pET21a-CJ_tn1, E. coli BL21 (DE3)/pET21a-CJ_tn2,respectively. These transformants were deposited at the Korean CultureCenter of Microorganisms under the provisions of the Budapest Treaty onMar. 20, 2017 with Accession Nos. KCCM11990P (E. coliBL21(DE3)/pET21a-CJ_ct1), KCCM11991P (E. coli BL21(DE3)/pET21a-CJ_ct2),KCCM11992P (E. coli BL, 21 (DE3)/pET21a-CJ_tn1), and KCCM11993P (E. coliBL21 (DE3)/pET21a-CJ_tn2), respectively.

Example 2: Production of Recombinant Enzymes

E. coli BL21(DE3)/pET21a-CJ_ct1, E. coli BL21(DE3)/pET21a-CJ_ct2, E.coli BL21(DE3)/pET21a-CJ_tn1, and E. coli BL21(DE3)/pET21a-CJ_tn2expressing each of the enzymes produced in Example 1 were seeded in aculture tube containing 5 ml of LB liquid medium, and then seed culturewas performed in a shaking incubator at 37° C. until absorbance at 600nm reached 2.0.

Each of the cultures obtained by the seed culture was seeded in aculture flask containing an LB liquid medium, and then main culture wasperformed. When absorbance at 600 nm reached 2.0, 1 mM IPTG was added toinduce expression and production of the recombinant enzymes. During theculture, a shaking speed was maintained at 180 rpm and a culturetemperature was maintained at 37° C. Each culture was centrifuged at8,000×g and 4° C. for 20 minutes to recover cells. The recovered cellswere washed with 50 mM Tris-HCl (pH 8.0) buffer twice and suspended inthe same buffer, followed by cell disruption using a sonicator. Celllysates were centrifuged at 13,000×g and 4° C. for 20 minutes to obtainonly supernatants. Each enzyme was purified therefrom using His-tagaffinity chromatography. The purified recombinant enzyme solution wasdialyzed against 50 mM Tris-HCl (pH 8.0) buffer, and used for reaction.

A molecular weight of each purified enzyme was examined by SDS-PAGE, andas a result, it was confirmed that CT1 (α-glucan phosphorylase) has amolecular weight of about 96 kDa, CT2 (phosphoglucomutase) has amolecular weight of about 53 kDa, and TN1(glucose-6-phosphate-isomerase) has a molecular weight of about 51 kDa(see FIG. 1).

Example 3: Examination of Fructose-6-Phosphate 4-Epimerase Activity ofTagatose-6-Phosphate Kinase

3-1. Production of Recombinant Expression Vector and RecombinantMicroorganism Comprising Tagatose-6-Phosphate Kinase Gene 1

To identify a novel heat-resistant fructose-6-phosphate-4-epimerase, anucleotide sequence expected as the enzyme was selected from anucleotide sequence of a thermophilic Anaerolinea thermophila, which isregistered in Genbank, and based on information of an amino acidsequence (SEQ ID NO: 1) and a nucleotide sequence (SEQ ID NO: 2) of themicroorganism, the gene was inserted into pBT7-C-His (Bioneer Corp.,)which is a recombinant vector expressible in E. coli to produce arecombinant expression vector designated as pBT7-C-His-an1. Theexpression vector was transformed into an E. coli BL21(DE3) strain by acommon transformation method [see Sambrook et al. 1989] to produce atransformant (transformed microorganism) designated as E. coliBL21(DE3)/pBT7-C-His-an1, and this transformant was deposited at theKorean Culture Center of Microorganisms (KCCM) under the provisions ofthe Budapest Treaty on Mar. 20, 2017 with Accession No. KCCM11996P (E.coli BL2l (DE3)/pBT7-C-His-an1).

3-2. Production of Recombinant Expression Vector and RecombinantMicroorganism Comprising Tagatose-6-Phosphate Kinase Gene 2

To provide fructose-6-phosphate-4-epimerase, genetic information oftagatose-6-phosphate kinase derived from Anaerolineae bacterium SG8_19or Dictyoglomus turgidum which is a thermophilic microorganism wasacquired to produce recombinant vectors expressible in E. coli andtransformed microorganisms.

In detail, a nucleotide sequence of tagatose-6-phosphate kinase wasselected from nucleotide sequences of Anaerolineae bacterium SG8_19 orDictyoglomus turgidum, which is registered in Genbank and KEGG (KyotoEncyclopedia of Genes and Genomes), and based on information of aminoacid sequences (SEQ ID NOS: 3 and 5) and nucleotide sequences (SEQ IDNOS: 4 and 6) of the two microorganisms, pBT7-C-His-CJ_AB_F6P4E andpBT7-C-His-CJ_DT_F6P4E which are recombinant vectors comprising thenucleotide sequence of the enzyme and being expressible in E. coli wereproduced (Bioneer Corp., Korea).

Each of the produced expression vectors was transformed into E. coliBL21(DE3) strain by a common transformation method [see: Sambrook et al.1989] to produce transformants (recombinant microorganisms) which weredesignated as E. coli BL21 (DE3)/pBT7-C-His-CJ_AB_F6P4E and E. coli BL21(DE3)/pBT7-C-His-CJ_DT_F6P4E, respectively. The transformants weredeposited at the Korean Culture Center of Microorganisms (KCCM) which isa depositary authority under the provisions of the Budapest Treaty withAccession Nos. KCCM12093P (date of deposit: Aug. 11, 2017) andKCCM12110P (date of deposit: Sep. 13, 2017), respectively.

3-3. Production of Recombinant Tagatose-6-Phosphate Kinase Enzyme

To produce recombinant enzymes, CJ_DT_F6P4E, CJ_AB_F6P4E, andCJ_AN1_F6P4E from the produced recombinant microorganisms, each of therecombinant microorganisms was seeded in a culture tube containing 5 mlof an LB liquid medium with ampicillin antibiotic, and then seed culturewas performed in a shaking incubator at 37° C. until absorbance at 600nm reached 2.0. Each of the cultures obtained by the seed culture wasseeded in a culture flask containing an LB liquid medium, and then mainculture was performed. When absorbance at 600 nm reached 2.0, 1 mM IPTG(isopropyl β-D-1-thiogalactopyranoside) was added to induce expressionand production of the recombinant enzyme. The seed culture and the mainculture were performed under conditions of 180 rpm and 37° C. Eachculture of the main culture was centrifuged at 8,000×g and 4° C. for 20minutes to recover cells. The recovered cells were washed with 25 mMTris-HCl (pH 7.0) buffer twice and suspended in the same buffer,followed by cell disruption using a sonicator. Each cell lysate wascentrifuged at 13,000×g and 4° C. for 20 minutes to take only asupernatant. The supernatant was purified using His-taq affinitychromatography, and 10 column volumes of 50 mM NaH₂PO₄ (pH 8.0) buffercontaining 20 mM imidazole and 300 mM NaCl was applied to removenon-specifically bound proteins. Next, 50 mM NaH₂PO₄ (pH 8.0) buffercontaining 250 mM imidazole and 300 mM NaCl was further applied toperform elution and purification. Dialysis was performed using 25 mMTris-HCl (pH 7.0) buffer to obtain CJ_DT_F6P4E, CJ_AB_F6P4E, andCJ_AN1_F6P4E which are purified enzymes for analysis of enzymecharacterization.

3-4. Analysis of Fructose-6-Phosphate 4-Epimerase Activity ofRecombinant Tagatose-6-Phosphate Kinase Enzyme

The fructose-6-phosphate-4-epimerization activities of the recombinanttagatose-6-phosphate kinase enzymes obtained in Example 3-3 wereanalyzed. In detail, 1% by weight of fructose-6-phosphate as a substratewas suspended in 25 mM Tris-HCl (pH 7.0) buffer, and each 1 unit/ml ofthe purified CJ_DT_F6P4E, CJ_AB_F6P4E, and CJ_AN1_F6P4E was addedthereto, and allowed to react at 60° C. for 1 hour. To remove phosphate,1 unit/ml of phosphatase (Alkaline phosphatase of NEB, Calf Intestinal)was added and allowed to react at 37° C. for 1 hour. Reaction productswere analyzed by HPLC, and HPLC analysis was performed under conditionsof using a SP0810(Shodex) column and applying a mobile phase (water) at80° C. and a flow rate of 1 ml/min, and resultants were analyzed using arefractive index detector.

As a result, it was confirmed that all of CJ_DT_F6P4E, CJ_AB_F6P4E, andCJ_AN1_F6P4E have the activity to convert fructose-6-phosphate intotagatose-6-phosphate (FIGS. 2 to 4).

Example 4: Identification of Tagatose-6-Phosphate Phosphatase Enzyme(D-Tagatose-6-Phosphate Phosphatase)

To perform production of tagatose from fructose-6-phosphate bysimultaneous complex enzyme reactions in the tagatose production pathwayof the present disclosure, tagatose-6-phosphate phosphatase which isable to exert the simultaneous enzyme reaction together withtagatose-6-phosphate kinase was identified.

4-1. Production of Recombinant Expression Vector and RecombinantMicroorganism Comprising Tagatose-6-Phosphate Phosphatase Gene

A nucleotide sequence (SEQ ID NO: 8, hereinafter, referred to as t4) andan amino acid sequence (SEQ ID NO: 7) expected as thetagatose-6-phosphate phosphatase were selected from a nucleotidesequence of Thermotoga maritima, which is registered in Genbank, andbased on the selected nucleotide sequence, a forward primer (SEQ ID NO:29) and a reverse primer (SEQ ID NO: 30) were designed and synthesized.Polymerase chain reaction (PCR) was performed using the primers andgenomic DNA of Thermotoga maritima as a template to amplify t4 gene. Theamplified gene of each enzyme was inserted into pET21a (Novagen) whichis a plasmid vector for expression in E. coli using restriction enzymesNdeI and XhoI, thereby producing a recombinant expression vector whichwas designated as pET21a-CJ_t4. The produced expression vector wastransformed into E. coli BL21(DE3) strain by heat shock transformation(Sambrook and Russell: Molecular cloning, 2001) to produce a recombinantmicroorganism, which was then used after being frozen and stored in 50%glycerol. The recombinant microorganism was designated as E. coliBL21(DE3)/pET21a-CJ_t4, and deposited at the Korean Culture Center ofMicroorganisms (KCCM) which is an International Depositary Authorityunder the provisions of the Budapest Treaty on Mar. 20, 2017 withAccession No. KCCM11994P.

4-2. Production of Recombinant Tagatose-6-Phosphate Phosphatase

E. coli BL21 (DE3)/pET21a-CJ_t4 was seeded in a culture tube containing5 ml of LB liquid medium and then seed culture was performed in ashaking incubator at 37° C. until absorbance at 600 nm reached 2.0. Theculture obtained by the seed culture was seeded in a culture flaskcontaining an LB liquid medium, and then main culture was performed.When absorbance at 600 nm reached 2.0, 1 mM IPTG was added to induceexpression and production of the recombinant enzymes. The seed cultureand the main culture were performed at a shaking speed of 180 rpm and37° C. The culture obtained by the main culture was centrifuged at8,000×g and 4° C. for 20 minutes to recover cells. The recovered cellswere washed with 50 mM Tris-HCl (pH 8.0) buffer twice and suspended inthe same buffer, followed by cell disruption using a sonicator. A celllysate was centrifuged at 13,000×g and 4° C. for 20 minutes to obtainonly a supernatant. The enzyme was purified therefrom using His-tagaffinity chromatography. The purified enzyme was used after dialysisagainst 50 mM Tris-HCl (pH 8.0) buffer, and the purified recombinantenzyme was designated as CJ_T4.

4-3. Analysis of Tagatose-6-Phosphate Phosphatase Activity of CJ_T4

To analyze activity of CJ_T4, tagatose-6-phosphate was suspended in 50mM Tris-HCl (pH 7.5) buffer, and 0.1 unit/ml of the purified CJ_T4 and10 mM MgCl2 were added thereto and allowed to react at 70° C. for 10minutes. Then, the reaction product was analyzed by HPLC. HPLC analysiswas performed under conditions of using a HPX-87H (Bio-Rad) column andapplying a mobile phase (water) at 60° C. and a flow rate of 0.6 ml/min,and tagatose and tagatose-6-phosphate were analyzed using a refractiveindex detector.

As a result, tagatose was produced in the reaction product. As a resultof performing the same reaction after adding CJ_T4 to phosphate andtagatose reactants, no tagatose was produced, indicating that CJ_T4 hasirreversible tagatose-6-phosphate phosphatase activity (FIG. 7).

Example 5: Production of Tagatose by Simultaneous Complex EnzymeReactions

To analyze the activity to produce tagatose from fructose-6-phosphate bycomplex enzymes, 0.1% (w/v) fructose-6-phosphate was added to a reactionsolution containing 1 unit/ml of CJ_t4 (Accession No. KCCM11994P), 1unit/ml of CJ_DT_F6P4E, 25 mM Tris-HCl (pH 7.0) buffer, and allowed toreact at 60° C. for 1 hour, and then HPLC was performed to analyze thereaction product. HPLC analysis was performed under conditions of usinga SP0810 (Shodex) column and applying a mobile phase (water) at 80° C.and a flow rate of 1 ml./min, and tagatose was detected using arefractive index detector.

As a result, tagatose production was observed, indicating that tagatosemay be produced from fructose-6-phosphate by simultaneous complex enzymereactions of tagatose-6-phosphate kinase and tagatose-6-phosphatephosphatase (FIG. 5).

Example 6: Production of Tagatose from Maltodextrin by SimultaneousComplex Enzyme Reactions

To analyze the activity to produce tagatose from maltodextrin by complexenzymes, 5% (w/v) maltodextrin was added to a reaction solutioncontaining 1 unit/ml of CT1, 1 unit/ml of CT2, 1 unit/ml of TN1, 1unit/ml of T4, 1 unit/ml of CJ_AN1_F6P4E, and 20 mM to 50 mM of sodiumphosphate (pH 7.0), and allowed to react at 60° C. for 1 hour, and thenHPLC was performed to analyze the reaction product. HPLC analysis wasperformed under conditions of using a SP0810 (Shodex) column andapplying a mobile phase (water) at 80° C. and a flow rate of 0.6 ml/min,and tagatose was detected using a refractive index detector.

As a result, it was confirmed that tagatose was produced frommaltodextrin by the complex enzyme reactions of added CT1, CT2, TN1, T4,and AN1 (FIG. 6).

Effect of the Invention

A method of producing tagatose according to the present disclosure iseconomical because of using glucose or starch as a raw material,accumulates no phosphate to achieve a high conversion rate, andcomprises a tagatose-6-phosphate phosphatase reaction which is anirreversible reaction pathway, thereby remarkably increasing aconversion rate into tagatose.

Further, tagatose may be produced from glucose or starch as a rawmaterial by complex enzyme reactions, and thus there are advantages thatthe method is simple and economical, and a yield is improved.

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11996P

Date of deposit: 20170320

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM12093P

Date of deposit: 20170811

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM12110P

Date of deposit: 20170913

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11990P

Date of deposit: 20170320

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11991P

Date of deposit: 20170320

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11992P

Date of deposit: 20170320

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11993P

Date of deposit: 20170320

International Depositary Authority: Korean Culture Center ofMicroorganisms (foreign)

Accession No: KCCM11994P

Date of deposit: 20170320

What is claimed is:
 1. A composition for producing tagatose-6-phosphate,comprising tagatose-6-phosphate kinase, a microorganism expressing thetagatose-6-phosphate kinase, or a culture of the microorganism.
 2. Thecomposition of claim 1, further comprising fructose-6-phosphate.
 3. Thecomposition of claim 1, wherein the tagatose-6-phosphate kinase consistsof an amino acid sequence of SEQ ID NO: 1, 3, or
 5. 4. A composition forproducing tagatose, comprising tagatose-6-phosphate kinase, amicroorganism expressing the tagatose-6-phosphate kinase, or a cultureof the microorganism; and tagatose-6-phosphate phosphatase, amicroorganism expressing the tagatose-6-phosphate phosphatase, or aculture of the microorganism.
 5. The composition of claim 4, furthercomprising fructose-6-phosphate.
 6. The composition of claim 4, furthercomprising glucose-6-phosphate isomerase, a microorganism expressing theglucose-6-phosphate isomerase, or a culture of the microorganism.
 7. Thecomposition of claim 6, further comprising phosphoglucomutase, amicroorganism expressing the phosphoglucomutase, or a culture of themicroorganism.
 8. The composition of claim 7, further comprisingα-glucan phosphorylase, starch phosphorylase, maltodextrinphosphorylase, or sucrose phosphorylase, a microorganism expressing theα-glucan phosphorylase, starch phosphorylase, maltodextrinphosphorylase, or sucrose phosphorylase, or a culture of themicroorganism.
 9. The composition of claim 6, further comprisingglucokinase, a microorganism expressing the glucokinase, or a culture ofthe microorganism.
 10. The composition of claim 9, further comprisingα-amylase, pullulanase, isoamylase, glucoamylase, or sucrase, amicroorganism expressing the α-amylase, pullulanase, isoamylase,glucoamylase, or sucrase, or a culture of the microorganism.
 11. Amethod of producing tagatose, comprising producing tagatose-6-phosphateby contacting fructose-6-phosphate with tagatose-6-phosphate kinase, amicroorganism expressing the tagatose-6-phosphate kinase, or a cultureof the microorganism.
 12. The method of claim 11, further comprisingproducing tagatose by contacting the produced tagatose-6-phosphate withtagatose-6-phosphate phosphatase, a microorganism expressing thetagatose-6-phosphate phosphatase, or a culture of the microorganism. 13.The method of claim 11 or 12, further comprising convertingglucose-6-phosphate into fructose-6-phosphate by contactingglucose-6-phosphate with glucose-6-phosphate-isomerase, a microorganismexpressing the glucose-6-phosphate-isomerase, or a culture of themicroorganism.
 14. The method of claim 13, further comprising convertingglucose-1-phosphate into glucose-6-phosphate by contactingglucose-1-phosphate with phosphoglucomutase, a microorganism expressingthe phosphoglucomutase, or a culture of the microorganism.
 15. Themethod of claim 14, further comprising converting starch, maltodextrin,or sucrose into glucose-1-phosphate by contacting starch, maltodextrin,sucrose, or a combination thereof with α-glucan phosphorylase, starchphosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase, amicroorganism expressing the α-glucan phosphorylase, starchphosphorylase, maltodextrin phosphorylase, or sucrose phosphorylase, ora culture of the microorganism.
 16. The method of claim 13, furthercomprising converting glucose into glucose-6-phosphate by contactingglucose with glucokinase, a microorganism expressing the glucokinase, ora culture of the microorganism.
 17. The method of claim 16, furthercomprising converting starch, maltodextrin or sucrose into glucose bycontacting starch, maltodextrin, sucrose, or a combination thereof withα-amylase, pullulanase, glucoamylase, sucrase, or isoamylase, amicroorganism expressing the α-amylase, pullulanase, glucoamylase,sucrase, or isoamylase, or a culture of the microorganism.
 18. Themethod of claim 11 or 12, wherein the contacting is performed at pH 5.0to 9.0, 40° C. to 80° C., and/or for 0.5 hours to 24 hours.
 19. Themethod of claim 11 or 12, wherein the tagatose-6-phosphate kinaseconsists of an amino acid sequence of SEQ ID NO: 1, 3, or 5, and thetagatose-6-phosphate phosphatase consists of an amino acid sequence ofSEQ ID NO:
 7. 20. A method of producing tagatose, comprising contacting(a) starch, maltodextrin, sucrose, or a combination thereof; with (b)(i) tagatose-6-phosphate phosphatase, (ii) tagatose-6-phosphate kinase,(iii) glucose-6-phosphate-isomerase, (iv) phosphoglucomutase orglucokinase, (v) phosphorylase, and (vi) one or more of α-amylase,pullulanase, isoamylase, glucoamylase, or sucrase; and (c) phosphate.